(A.) In a collaboration with Dr. Joachim Heberle in Germany, we have combined time-resolved optical and FTIR spectroscopies to capitalize on the different abilities of each method to reveal the changes in protein conformation and liganding that accompany spectrally defined transitions during the bacteriorhodopsin (BR) photocycle. The specific goal is to examine native purple membranes (PM) in comparison to Triton-treated and various stages of lipid-reconstituted preparations to look for strong correlations in the kinetics of proton release and uptake with well-defined intermediate steps in the photocycle. All preparations of material are made in our laboratory and the same preparation is examined optically by us and by Heberle in Germany using FTIR. Then both data sets are combined in one large matrix and examined by us using singular value decomposition (SVD). The power of this combined approach was demonstrated by the fact that the optical data alone could be fit to 6 exponentials and the FTIR data alone to 5 exponentials, but the combined data set revealed 7 exponential transitions. This is because there is different information in the two data sets and the combination contains more information than either set alone. The simultaneous SVD deconvolution reveals changes in protein conformation and liganding for each isolated step in the photocycle. The 7-exponential solution split the L->M transition normally seen at 100 us into two steps at 59 and 158 us. It also resolved a step, we originally saw as an M->O step into an M->N and N->O steps. The combined Triton-treated data revealed much slower kinetics and three L- >M transitions, as well as three M->BR transitions, and an absence of the M-fast intermediate. (B.) Our previous work has shown that the neutral lipid, squalene is essential for formation of the M-fast (Mf) intermediate. We find that the neutral solvent, decane, can convert the M-slow (Ms) intermediate into Mf. We now have a variety of ways to alter the relative amounts of Mf and Ms in the BR photocycle: 1. Triton, high pH, high energy actinic light and membrane potential all convert Mf to Ms. 2. Native lipids, particularly squalene with phosphatidyl glycerophosphate (PGP), or decane convert Ms to Mf. The ability to prepare samples with widely different ratios of Mf/Ms should help in elucidating the protein conformational forms that characterize Mf and Ms and perhaps their individual roles in energy transduction. (C.) Use of site-directed mutants to study the importance of a suspected squalene-PGP-acidic aminoacid interaction for Mf activity. Our finding that reconstitution of native photocycle activity by addition of native lipids to Triton-treated PM requires either high salt or pH titration with an apparent pK of ~5, suggests such an interaction. We are using specific mutants where acidic residues exist closely to the lipids of the membrane to test this hypothesis, specifically, D38N, D36N, D102N/D104N, and D36N/D38N/D102N. A common feature is that the loss of D36 and D38 does result in a dramatic decrease in the amount of Mf and in the buildup of a very slow form of M. (D.) A paper was published (JACS, (1999) 121:1385-1386) with Ad Bax showing that magnetically oriented PM membranes can be used to provide the required weak degree macromolecular alignment in strong magnetic fields to allow the NMR measurement of dipolar couplings of certain added soluble proteins. - bionergetics, spectral deconvolution, SVD, time-resolved FTIR and optical spectra